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Seasonal and pandemic influenza virus infections not only pose a serious threat to human health but also cause tremendous economic losses and social burdens. However, due to the inherent high variability of influenza virus RNA genomes, the existing anti-influenza virus drugs have been frequently faced with the clinical issue of emerging drug-resistant mutants. Therefore, there is an urgent need to develop efficient and broad-spectrum antiviral agents against wild-type and drug-resistant mutant strains. Phenotypic screening has been widely employed as a reliable strategy to evaluate antiviral efficacy of novel agents independent of their modes of action, either directly targeting viral proteins or regulating cellular factors involved in the virus life cycle. Here, from the point of view of medicinal chemistry, we review the research progress of phenotypic screening strategies by focusing direct acting antivirals against influenza virus. It could provide scientific insights into discovery of a distinctive class of therapeutic candidates that ensure high efficiency but low cytotoxicity, and address issues from circulation of drug-resistant influenza viruses in the future., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (This journal is © The Royal Society of Chemistry.)